We recently demonstrated that soon after Bacillus anthracis spores are delivered into mouse lungs significant quantities of these spores are found inside the lung epithelial cells. Here we hypothesize that this intracellular presence plays an important role in B. anthracis pathogenesis. Specifically, we propose a model where spores enter lung epithelial cells at the early stages of infection and they either germinate into vegetative bacilli and are translocated across the epithelial cells, or, they persist inside them as spores, i.e., intracellular germination is a prerequisite to switching on a "translocation pathway". The crucial importance of dissemination and germination in inhalational anthrax is well established. Persistence of spores in the lung has also been noted for decades. However, the mechanisms permitting persistence, germination and dissemination in vivo are poorly understood or unknown. Our hypothesis that the intracellular environment of the lung epithelial cells provides a venue for all these events is a novel concept and provides an alternative pathway to the classical model of macrophage uptake and dissemination. This is made more compelling by recent evidence that B. anthracis bacilli are susceptible to phagocytic killing by macrophages both in vitro and in vivo. Therefore, this new model has significant advantages for the bacterium in terms of survivability. Consequently results from this application will have a significant impact on understanding the pathogenic mechanisms of B. anthracis. From a broader point of view, dissemination and persistence are two important issues concerning many microbial infections. Despite the importance, much remains to be elucidated including what roles the lung epithelium can play during an infection. Therefore, this application will have broad implications to bacterial pathogenesis in general as well as enhancing our understanding of the function of the lung epithelium under microbial challenge. Our new model is further supported by the following data. B. anthracis spores are internalized by cultured epithelial cell lines and primary lung epithelial cells. B. anthracis can survive inside lung epithelial cells and can cross an in vitro model of the alveolar epithelium from the apical to the basolateral side via a transcellular route. Only germinated spores/vegetative bacilli are recovered from the basolateral side of the epithelium even under assay conditions that do not permit extracellular germination of spores, suggesting that germination occurs inside epithelial cells and is a prerequisite for exiting these cells. In this application, we propose three specific aims to determine if lung epithelial cells are a site for dissemination, persistence and germination in vivo. In future studies, we will investigate the molecular basis underlying these events and the dynamic balances between these events. PUBLIC HEALTH RELEVANCE: How Bacillus anthracis, the causative agent of anthrax, spreads in the host body and persists in the lung is not well understood. Our studies suggest that the lung epithelium may play a critical role in these processes. Investigation of this possibility will improve the current understanding of how this bacterium causes disease and may reveal new therapeutic strategies.